The NF-KB Pathway and Endocrine Therapy Resistance in Breast Cancer

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Endocrine-Related P Khongthong et al. Nuclear factor kappa B and 26:6 R369–R380 Cancer breast cancer REVIEW The NF-KB pathway and endocrine therapy resistance in breast cancer

Phungern Khongthong, Antonia K Roseweir and Joanne Edwards

Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, College of MVLS, University of Glasgow, Glasgow, UK

Correspondence should be addressed to J Edwards: [email protected]

Abstract

Breast cancer is a heterogeneous disease, which over time acquires various adaptive Key Words changes leading to more aggressive biological characteristics and development of ff NF-KB treatment resistance. Several mechanisms of resistance have been established; however, ff endocrine therapy due to the complexity of oestrogen receptor (ER) signalling and its crosstalk with other resistance signalling networks, various areas still need to be investigated. This article focusses ff breast cancer on the role of nuclear factor kappa B (NF-KB) as a key link between inflammation and cancer and addresses its emerging role as a key player in endocrine therapy resistance. Understanding the precise mechanism of NF-KB-driven endocrine therapy resistance provides a possible opportunity for therapeutic intervention. Endocrine-Related Cancer (2019) 26, R369–R380

Introduction

Oestrogen receptor α-positive (ER+) breast cancer respond to endocrine therapies as a result of either de constitutes more than 70% of all breast cancers novo or acquired resistance (Liu et al. 2017). (Cardoso et al. 2012). Both early and metastatic disease Many comprehensive reviews (Riggins et al. 2007, are treated effectively with endocrine therapies, which Clarke et al. 2009, Zhao & Ramaswamy 2014, Liu et al. downregulate oestrogen receptor (ER) signalling, leading 2017, AlFakeeh & Brezden-Masley 2018, Masuda et al. to tumour growth inhibition (Cardoso et al. 2012). The 2018) summarize the mechanisms of endocrine therapy three main groups of endocrine therapy are selective resistance, including (i) the loss of ER expression or oestrogen receptor modulators, including tamoxifen mutation of the ER that causes constitutive activation and raloxifene, which acts as an oestrogen antagonist regardless of oestrogen; (ii) the amplification and to bind to ER and further recruit transcriptional upregulation of ER co-activators such as amplified co-repressor instead of co-activators, leading to in breast 1 (AIB1, also known as steroid receptor inhibition of tumour growth (Legha 1988); selective co-activator-3 (SRC3)), that can increase the activity oestrogen receptor downregulators (SERDs), including of ERs; (iii) the upregulation of alternative oncogenic fulvestrant, which prevents ER dimerization and induce pathways such as phosphatidylinositol 3-kinase/a serine- its degradation (Wardell et al. 2011); and aromatase threonine-specific protein kinase/mammalian target inhibitors (AIs), including anastrozole, letrozole and of rapamycin (PI3K/AKT/mTOR) pathway resulting in exemestane, which inhibit the enzyme aromatase increased activity of protein kinase pathways; (iv) the resulting in reduction of oestrogen levels through the amplification and overexpression of gene regions that blockade of testosterone conversion to oestrogens both encode oncogenic proteins and transcription factors to in the tumour and peripheral tissue (Baum et al. 2003). promote cancer cell survival, invasion and metastasis; However, a significant number of the patient fail to and (v) the deregulation of the proteins that control cell

-19-0087 Endocrine-Related P Khongthong et al. Nuclear factor kappa B and 26:6 R370 Cancer breast cancer cycle machinery such as the cyclin-dependent kinases their target genes through the recruitment of co-activators (CDKs), resulting in dysregulated cellular proliferation. and co-repressors (Zhang et al. 2017). The potent Among these mechanisms, mTOR deregulation is transcription activation domain (TAD) is found only in considered clinically relevant and the mTOR inhibitor p65, c-Rel and RelB. Due to the lack of TADs, dimers of everolimus plus exemestane is included as an option for p50 or p52 may mediate only transcriptional repression ER-positive advanced disease; however, this combination (Zhang et al. 2017). failed to improve overall survival in clinical trials (Baselga In unstimulated cells, homo- or heterodimers of et al. 2012). In addition, due to double improvement in NF-KB are bound to their inhibitors and the IκB proteins progression-free survival, palbociclib, a highly selective are sequestered in the cytoplasm (Zhang et al. 2017). inhibitor of CDK4/6 kinases, in combination with Upon activation, NF-KB translocates to the nucleus letrozole had recently approved as first-line endocrine to interact with κB site to induce transcription of the therapy for postmenopausal ER-positive, HER2-negative target genes (Zhang et al. 2017). The most frequently advance breast cancer (Finn et al. 2015). Despite the recent recognized NF-KB pathways are the canonical and non- advances in therapeutic approach, metastatic breast canonical pathway (Zhang et al. 2017). The canonical cancer is incurable, and finally will develop treatment NF-KB pathway is activated by pro-inflammatory resistance, further increasing the complexity of molecular cytokines such as TNF-α and interleukin-1 (IL-1), interactions. Therefore, identifying the novel driving T- and B-cell mitogens, bacterial liposaccharide (LPS), factors that modulate oestrogen signalling and other viral proteins, and physical and chemical stress important pathways still need to be addressed. (Karin & Ben-Neriah 2000, Hoesel & Schmid 2013) (Fig. 1). Nuclear factor kappa B (NF-KB) is a key transcription A first step in the canonical pathway is the activation factor that links inflammation with cancer and is of transforming growth factor-β (TGF-β)-activated kinase demonstrated as being involved in the tumorigenesis of 1 (TAK1), which further activates a trimeric IkB kinase breast cancer and endocrine therapy resistance. However, (IKK) complex that is composed of regulatory (NF-KB the molecular mechanisms of how NF-KB contributes to essential modulator (NEMO or IKKγ)) and catalytic (IKKα endocrine therapy resistance are still unclear. This article and IKKβ) subunits (Zhang et al. 2017). The IKK complex will summarize the currently available evidence that then phosphorylates IκB at specific serine residues, supports the promising role of NF-KB pathway in the which results in polyubiquitination and subsequent mechanism of endocrine therapy resistance, in order to proteasomal degradation of IκB, followed by nuclear elucidate NF-KB as a potential novel target for overcoming translocation of NF-KB, mainly the p50/p65 heterodimer this resistance. (Zhang et al. 2017). The heterodimer then binds to the κB site and activates numerous genes that involve in inflammatory and immune responses, including cytokines, chemokines, inflammatory mediators, NF-KB signalling adhesion molecules and apoptosis inhibitors (Hoesel The NF-KB family of inducible transcription factors are & Schmid 2013, Lim et al. 2016, Zhang et al. 2017). composed of five members, including p50, p52, p65 In contrast to the canonical pathway that responds (RelA), RelB and c-Rel, all of which share an N-terminal rapidly to signals from the diverse receptors, the non- Rel homology domain (RHD) (Zhang et al. 2017). RHD canonical pathway specifically responds to a small group contains a nuclear localization sequence, and is responsible of receptors, including lymphotoxin-β receptor (LTβR), for sequence-specific DNA binding, dimerization and B-cell activating factor belonging to TNF family receptor interaction with ankyrin repeat motifs, which are (BAFFR), CD40 and receptor activator for NF-KB (RANK), present in IκB inhibitory proteins (Zhang et al. 2017). as summarized in recent comprehensive review (Sun The ‘inhibitor of κB’ (IκB) proteins include IκBα, IκBβ, 2012, 2017). Upon binding to its specific ligand, these IκBγ, IκBϵ, BcL-3, the precursors p105 and p100, and the receptors activate the kinase NF-KB-inducing kinase Drosophila protein Cactus (Zhang et al. 2017). Among (NIK), which, in turn, phosphorylates and activates them, IκBα, IκBβ and IκBϵ are the most important IKKα. Activated IKKα then phosphorylates p100/RelB regulators of NF-KB. NF-KB dimers bind to κB sites within heterodimer at the carboxyterminal serine residues of the promoters or enhancers of target genes, which bare p100, subsequent to proteasomal degradation of the C- 5’-GGGRNWYYCC-3’ (N, any base; R, purine; W, adenine terminal IκB-like structure of p100, resulting in processing or thymine; Y, pyrimidine), and regulate transcription of of p100 to p52 and nuclear translocation of p52/RelB

CanonicalNF-kB pathwayNon-canonicalNF-kB pathway

TNFα, IL1 LT,CD40L BCR TNFR LTβR,CD40 Cytoplasm

TAK1 NIK Figure 1 IKKγ p The NF-KB pathway. The canonical pathway is p P P IKKα IKKα IKKα IKKβ induced by TNFα, IL1 and various other stimuli, and is an IKKβ-dependent cascade. Activation of this p p Ub p RelB cascade leads to the phosphorylation of IkBα p p100 IκBα resulting in degradation by the proteasome. This p65 p50 Ub releases the NF-KB complex and allows it to translocate to the nucleus. The non-canonical pathway is induced by specific stimuli such as lymphotoxin α and is an IKKα-dependent cascade. Activation of this cascade results in phosphorylation of NIK, followed by phosphorylation of IKKα and subsequent phosphorylation of the p100 NF-KB Nucleus subunit. This subunit is then processed to p52, which leads to the activation of the p52-RelB p65 p50 RelB p100 heterodimer. The heterodimer can then translocate to the nucleus where it controls the transcriptional processing of its target genes. A full colour version of this figure is available athttps://doi.org/10.1530/ NF-kBtargetgene ERC-19-0087.

(Sun 2012, 2017) (Fig. 2). Activated p52/RelB heterodimer (Nakshatri et al. 1997, Sovak et al. 1997, Nakshatri & then binds to the specificκ B enhancer and subsequently Goulet 2002, Zhou et al. 2005b). activates transcription of its target genes that are involved With regard to endocrine therapy resistance, studies in many biological function, including secondary have demonstrated that (i) increasing p65 expression lymphoid organogenesis, B-cell maturation and was seen in MCF7/LCC9 cell (ER-positive, oestrogen survival, dendritic cell maturation and differentiation independent for growth and anti-oestrogen cross-resistant) of osteoclasts (Sun 2017). Together, it is not surprising and MCF7/RR cells (ER-positive, oestrogen-independent that NF-KB serves as a key player in the immune system, for growth, tamoxifen resistant and fulvestrant sensitive), cell survival, differentiation, proliferation and apoptosis. compared with MCF-7 control cells (Nehra et al. 2010); Moreover, a number of studies have confirmed NF-KB (ii) increasing basal transcriptional activity of NF-KB was is key for crosstalk between inflammation and cancer shown in MCF7/RR cells compared with MCF-7 control (Hoesel & Schmid 2013, Zhang et al. 2017). cells (Nehra et al. 2010); (iii) using the NF-KB-inhibitor parthenolide resulted in decreased cell proliferation, and significant inhibition of NF-KB-dependent transcription in both resistance cell lines, compared to MCF-7 cells (Nehra NF-KB signalling in breast cancer et al. 2010); (iv) enhanced NF-KB and AP-1 transcriptional A genetic analysis using a mouse model demonstrated activity was found in tamoxifen-resistant breast cancer that NF-KB plays a critical role in regulating proliferation cell and NF-KB and AP-1 regulated gene expression was of the mammary epithelial cells during pregnancy, which suppressed after treatment with the NF-KB inhibitor, is controlled by a linear cascade of at least six components parthenolide, or the proteasome inhibitor, bortezomib of the NF-KB pathway, including RANKL, RANK, IKKα, (Zhou et al. 2007); and (v) high DNA-binding activity of IkBα, p50/p65 and CyclinD1 (Cao et al. 2001). In the p50 subunit of NF-KB is a potential prognostic marker addition, a growing body of literature demonstrated to identify a high-risk subset of ER-positive primary breast that deregulation or constitutive activation of the NF-KB cancer, which are found to have early relapse disease pathway is involved in tumorigenesis of breast cancer despite adjuvant treatment with tamoxifen (Zhou et al. and is associated with the hormone-independent setting 2005a). Collectively, these finding support a crucial role

Macrophage

EGF Estradiol TNFα

Tcell TNFα FasL ER ERER ER ER ERER TNFR EGFR ERPI3K TNFR PD-1 FasR PI3K ER stress EnR Cytoplasm PD-L1 Mitochondria PKCθ Caspase-8 TAK1 AKT ProteinC BCL2 Ub IRE IKKγ p Unfold protein P P P IKKα IKKβ Caspase-9 IKKα IKKα FOXO3a XBP1 mTOR CSN5 Ub IκBα Increased p50 Cleaved UPRsignaling Nuclear p65 exportation caspase-3

p27Kip1 P CoA P Autophagosome SARC3 FOXO3aERER ERER ER Cyclin D ac ac IKKα Cyclin D FOX1 ERER ERER Apoptosis CSN5 Poised ER enhancer ac me me p65 ac ac IKKα E2F E2F Cyclin D CBP FOX1 ERER ERER p65 p50 p65 BIRC3 cREL p50 me Lalent ER enhancer me p50 NkBpromotor XBP1 CDK4/6 Cyclin D BCL2 Newsetsof Nucleolus synergistic E2F E2F Rb gene

G1 S A B C D E F G

Growth factor signalingCell De-suppressing of cREL cyclemachinery ReshapeERtranscriptome NF-kBpathway Immune surveillance Apoptosispathway UPRsignaling

Crosstalkbetween NF-kBand ER signaling NF-kBpathway andother crosstalkpathway

Figure 2 Crosstalk between NF-KB, ER signalling and other oncogenic pathway in endocrine resistance breast cancer. (A) PKCθ activates AKT, which in turn induces nuclear exportation of FOXO3a, resulting in decreased ERα transcriptional activity, leading to re-activation of c-Rel activity, followed by proliferation and an invasive phenotype transformation of breast cancer. (B) Upon activation with PI3K, IKKα phosphorylates both ER and SRC3 to further increase their transcriptional activity and acetylates E2F1 to increase the expression of E2F1-regulated genes, followed by increased cell cycle progression. (C) Upon co-stimulation with E2+TNFα, NF-KB and the Forkhead protein FoxA1 combine and locate new ER-binding sites named as a latent enhancer that controls up- or downregulation of a new set of synergistically regulated gene. (D) The NF-KB pathway as described in Fig. 1. (E) Upon TNF-α stimulation, NF κB p65 subunit bind to CSN5 promoter and promotes its transcription which subsequently induces its binding to PD-L1 and further deubiquitinates and stabilizes this protein, resulting in prevention of immune surveillance. (F) NF-KB is involved in the apoptotic machinery through transcriptional deregulation of pro-apoptotic (BAX, BAK, BAD and BCLXS) or anti-apoptotic proteins (BCL2, BCLXL and BCLW). NF-KB inhibits apoptosis in resistant cell through increased BCL2 expression, which further alters the BCL2:BAX ratio and inhibits apoptosis and modulates caspase 8 to further affect BCL2 resulting in inhibited apoptosis. (G) Accumulation of unfolded or misfolded proteins is detected by endoplasmic reticular transmembrane receptors such as inositol-requiring protein-1 (IRE1), which then activates XBP1 to enhance the unfolded protein response, which then promote phagocytosis. A full colour version of this figure is available athttps://doi.org/10.1530/ERC-19-0087 . for the NF-KB pathway in endocrine therapy resistance of or co-repressor, which will then alter ER transcriptional breast cancer. activity (Park et al. 2005). Similar to ER, NF-KB has been reported to have a role as a downstream effector for the growth factor pathway, which is recognized to be involved in both ligand-dependent and non-ligand-dependent ER Crosstalk between NF-KB and ER signalling in activation, leading to resistance to a wide range of anti- endocrine therapy resistance breast cancer oestrogen drugs (Zhou et al. 2005a, Sas et al. 2012, Frasor Many comprehensive reviews have demonstrated that et al. 2015). Moreover, NF-KB plays a key role in the anti- multiple mechanisms are involved in the crosstalk apoptotic pathway and immune surveillance mechanisms, between NF-KB and ER. NF-KB directly interacts with the which are demonstrated to be part of endocrine resistance DNA-binding function of ER through several mechanisms, (Hu et al. 2015, Lim et al. 2016). In addition, NF-KB such as collaboration with FOXA1 to enhance latent repression of ER activity has also been demonstrated. ER-binding sites, and induce translation of their The NFκB subunit, RelB, induces the expression of the synergistic genes (Franco et al. 2015). In addition, NF-KB zinc finger repressor B-lymphocyte-induced maturation influences ER through association with its ER co-activator protein (BLIMP1), which can bind to the ER promoter

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0087 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 08:06:07PM via free access Endocrine-Related P Khongthong et al. Nuclear factor kappa B and 26:6 R373 Cancer breast cancer region and repress ER transcription (Wang et al. 2009). 2014). These mutations enhance stabilization of the Currently, increasing evidence demonstrates that NF-KB is active conformation of ER, leading to constitutive a crucial part in the complexity of the endocrine resistance activation and AI resistance (Barone et al. 2010, setting within breast cancer. Tryfonidis et al. 2016). It has been demonstrated that there is a synergistic effect between NF-KB and ER that enhances expression of oncogenic proteins as described NF-KB and ERS1 mutation in endocrine therapy in the following section, it is not surprising therefore resistance breast cancer that ESR1 mutations might indirectly crosstalk to NF-KB Oestrogens (mainly 17β-estradiol) are a group of steroid pathway through the promotion of ER activity. Due to hormones that control cell growth and differentiation. limited current evidence that reports a role for NF-KB Oestrogen binds to ER that exists in two isoforms, in ER-positive breast cancer, patients carrying ESR1 ERα and ERβ, which are encoded by two particular genes, mutations still need further study. ESR1 and ESR2, respectively. While ERα plays a key role in regulating genes transcription that is involved in the NF-KB and FOXA1 is required for TNFα to modulate mitogenic pathway in breast cancer, the role of ERβ as a latent ER-binding sites to reshape the breast cancer transcription factor remains unclear. cell transcriptome ER regulates transcription of its target gene through classical genomic, non-classical genomic and non- Increasing evidence reveals that inflammation plays a genomic pathways (Shou et al. 2004, Sas et al. 2012, Zhao key role in maintaining the tumour microenvironment & Ramaswamy 2014). The classical genomic pathway and promotes a more aggressive phenotype of ER-positive initiates with the binding of oestrogen to ER resulting in tumour (Baumgarten & Frasor 2012). In contrast to the release of heat shock protein (hsp) 90, followed by the potent mitogenic effect of oestrogens in breast ER dimerization and nuclear translocation, where it can cancer cells, pro-inflammatory signalling in the tumour directly bind to the oestrogen response elements (EREs) microenvironment can either promote or inhibit tumour in target genes (Shou et al. 2004, Sas et al. 2012, Zhao proliferation, depending on the particular context & Ramaswamy 2014). In contrast, in the non-classical (Ben-Neriah & Karin 2011). NF-KB is a crucial transcription genomic pathway, ER dimers do not bind to the EREs factors for pro-inflammatory signalling and is required directly, but associate with other transcription factors for TNFα to promote crosstalk between mitogenic and prior to binding (Shou et al. 2004, Sas et al. 2012, Zhao pro-inflammatory signalling pathways in breast cancer & Ramaswamy 2014). Whereas, in the non-genomic cells (Franco et al. 2015). pathway, upon binding to oestrogen, ER activates other By using chromatin immunoprecipitation (ChIP) pathways, including the mitogen-activated kinase for ER and the p65 subunit of NF-KB, three distinct sets pathway (MAPK) and the PI3K-AKT-mTOR pathway, to of ERα-binding sites or enhancers were identified upon promote cell survival, proliferation and angiogenesis co-treatment with E2+TNFα, with a high prevalence of (Shou et al. 2004, Sas et al. 2012, Zhao & Ramaswamy p65 and ESR1 motifs found to be gained at ERα-binding 2014). Furthermore, activation of ER by growth factor site, indicating functional interactions between ER signalling factors such as IGF and epidermal growth and NF-KB across the genome (Franco et al. 2015). In factor receptors (EGFRs) can mediate ER activation by addition, upon co-stimulation with E2+TNFα, NF-KB and inducing its phosphorylation, which can then translocate the Forkhead protein, FoxA1 (a well-established pioneer to the nucleus to bind to EREs, independent of oestrogen factor for ER), combine and locate new ER-binding sites binding. Several mechanisms of endocrine therapy that are found in less-accessible regions on the genome resistance have been identified, many of which are related and are not recognized by the classical oestrogen-induced to the complex function of ER and its crosstalk with the ER cistrome (Franco et al. 2015). Addition of TNFα to E2 other cellular pathway as described above. Therefore alters extensive gene expression programme results in the altered ER function, which results from ESR1 mutation up- or downregulation of a new set of genes, which were or ER loss of function, has been established as one of the unchanged by either E2 or TNAα alone. The new sets of major resistance mechanisms (Barone et al. 2010). gene that arise from this transcriptome are involved in ESR1 mutations rise from 2% in the primary tumour variety of cellular processes, including cellular signalling, to nearly 30 % in metastatic disease and the endocrine- protein and lipid metabolism, proliferation, programmed resistant setting (Barone et al. 2010, Jeselsohn et al. cell death and apoptosis (Franco et al. 2015).

Moreover, the Gene Expression-Based Outcome (Hanahan & Weinberg 2011). The apoptotic machinery for Breast Cancer Online tool was used to examine the consists of both upstream regulators and downstream association between the unique E2 + TNFα-regulated effectors (Wong 2011). The regulators are divided into two transcriptome and clinical outcome (Franco et al. 2015). distinct molecular pathways: the intrinsic and extrinsic The result demonstrated that this group of gene sets pathway (Wong 2011). The intrinsic or mitochondria- are influential predictors of clinical outcomes; however, dependent pathway is activated by various types of good or poor outcomes depend on the particular breast intracellular signals and relies upon pro-apoptotic or anti- cancer types (Franco et al. 2015). For example, high apoptotic molecules that are released from mitochondria expression levels of E2+TNFα de novo and synergistically such as cytochrome C, apoptosis-inducing factor, second upregulated gene sets are associated with high overall mitochondria-derived activator of caspase (Smac), direct survival in patient with ER-positive, lymph node-positive IAP-binding protein with low pI (DIABLO) and the Bcl-2 tumours and are associated with low overall survival family of proteins, which are divided mainly into two in patients with untreated tumour (Franco et al. 2015). groups, namely the pro-apoptotic proteins (e.g. BAX, Furthermore, high expression levels of E2+TNFα de novo BAK, BAD and BCLXs) and the anti-apoptotic proteins and synergistically downregulated gene sets are associated (e.g. BCL2, BCLXL and BCLW) (Wong 2011). The extrinsic with low overall survival in patient with ER-positive and pathway is activated by the binding of death ligands to lymph node-negative tumours (Franco et al. 2015). their cell-surface death receptor such as the Fas receptor Collectively, these results indicate that NF-KB is a key and the type 1 TNF receptor (TNFR1) (Wong 2011). Their link between pro-inflammatory and mitogenic signalling, ligands are called Fas ligand and TNF, respectively. The leading to creation of latent ER-binding site that reshape downstream effectors involve the activation of a series of patterns of gene expression, and further contribute caspases, including caspase 9 and caspase 8, which are the to varied cellular responses and clinical outcomes in upstream caspases for the intrinsic and extrinsic pathway, particular breast cancer subtypes. respectively (Wong 2011). These two pathways converge on caspase 3, which then induces cleavage of many proteins such as protein kinases, DNA repair protein, NF-KB and the PKCθ/AKT/FOXO3a axis induce invasive cytoskeletal proteins and inhibitory subunits of the transformation of breast cancer through repressing endonuclease family (Wong 2011). Following this step, of ER transcription the cell is progressively disassembled and then consumed The serine/threonine protein kinase C θ (PKCθ) has been by phagocytes (Wong 2011). reported to regulate c-Rel-driven mammary tumour in Increasing evidence reveals that NF-KB is involved mouse models (Guo & Sonenshein 2004). In the normal in the apoptotic machinery through transcriptional mammary gland, FOXO3a binds to the Forkhead box dysregulation of pro-apoptotic or anti-apoptotic proteins elements in the ER promoter and maintains a normal such as TRAF proteins, the BCL2 family of proteins, epithelial cell phenotype via induced transcription of its p53 and inhibitor of apoptosis proteins (IAPs), resulting target genes p27Kip1 (a member of the universal CDK in reduced apoptosis and promoting survival in many inhibitor) and ERα, which in turn represses c-Rel activity cancers, including breast cancer (Baldwin 2001, Clarke (Guo & Sonenshein 2004). In the invasive phenotype et al. 2009, Schmitz et al. 2018). The following section will transformation of breast cancer, PKCθ activates AKT, detail the major mechanisms that play a critical role in which in turn induces nuclear exportation of FOXO3a, governing apoptosis via the NF-KB pathway. resulting in decreased ERα transcription activity, leading to re-activation of c-Rel activity, followed by proliferation NF-KB and ER synergistically enhance expression of and invasive phenotype transformation of breast cancer the anti-apoptotic gene, BIRC3 (Guo & Sonenshein 2004). Although much of literature has demonstrated trans- repression between ER and NF-KB, prominent positive crosstalk has also been identified via genome-wide NF-KB mediates anti-oestrogen resistance transcriptional analysis of ER and NF-KB in hormone- through inhibition of the apoptotic pathway positive breast cancer cell lines. Treatment with oestrogen Development of a various strategies to inhibit or prevent enhances TNFα activity on nearly 15% of TNFα-upregulated apoptosis is one of the most important hallmarks of cancer genes, which includes Baculoviral IAP repeat containing 3

(BIRC3)/cellular inhibitor of apoptosis protein 2 (cIAP-2) protein responses that drive endoplasmic reticulum (Frasor et al. 2009). Importantly, treatment with oestrogen stress to maintain cell homeostasis (Tyson et al. 2011). in combination with TNFα enhanced the upregulation Overexpression of a spliced form, XBP1(S), in ER-positive of BIRC3 (Frasor et al. 2009). BIRC3 is an important breast cancer cells induces oestrogen-independent growth anti-apoptotic gene and is known to be upregulated and resistance to anti-oestrogens (Gomez et al. 2007, by NF-KB in response to TNFα stimulation (Frasor et al. 2007). Addition of the NF-KB inhibitor parthenolide 2009). This finding suggests a synergistic role between to 4-hydroxytamoxifen (4-OHT) strongly sensitized ER and NF-KB in hormone-positive breast cancer that XBP1-overexpressing MCF-7 cells to 4-OHT. Similar might promote an aggressive phenotype via activation to this result, p65 siRNA inhibited growth in XBP1 of anti-apoptotic genes. overexpressing MCF-7 cells (Gomez et al. 2007). Furthermore, highly sensitivity to 4-OHT was found in p65-depleted XBP1-overexpressing MCF-7 cells (Gomez NF-KB inhibits mitochondrial membrane et al. 2007). In addition, increase annexin V staining permeability, and reduces caspase-dependent (apoptosis marker) and increased level of LC3II and apoptotic cell death through caspase 8 modulation p62 (autophagy marker) were observed in p65-depleted and BCL2 expression XBP1-overexpressing MCF-7 cells treated with 4-OHT, Evidence that confirms an anti-apoptotic role of NF-KB indicating that XBP1-NF-KB signalling mediates anti- in endocrine therapy resistance is provided by a study oestrogen resistance through blocking apoptosis and using anti-oestrogen-resistant cells line (MCF7/RR and autophagy (Gomez et al. 2007). Furthermore, the average MCF7/LCC9), which demonstrates both high basal p65 tumour volume in XBP1-overexpressing xenografts expression and transcriptional activity of NF-KB compared treated with parthenolide were significantly less than with oestrogen-sensitive MCF-7 cells (Nehra et al. 2010). those in the control group (Gomez et al. 2007). These Inhibition of NF-KB, by either a small-molecule inhibitor results strongly confirm that XBP1-mediated anti- of NF-KB or a mutant IκB (IκBSR), synergistically sensitizes oestrogen resistance is NF-KB dependent. However, the both resistant cell lines to 4-hydroxytamoxifen (4HT), mechanism that is accountable for the XBP1 regulation which leads to enhanced cancer cell apoptosis (Nehra of p65/RelA remains unknown. et al. 2010). The study demonstrated that NF-KB inhibits apoptosis in resistant cells through increased BCL2 expression, which further alters the BCL2:BAX ratio, NF-KB prevents immune surveillance in consequently inhibiting mitochondrial permeability to endocrine-resistant breast cancer decrease cytochrome c excretion, resulting in inactivation of caspase activities and preventing apoptosis (Nehra Programmed cell death ligand 1 (PD-L1) frequently et al. 2010). The study also reported that NF-KB inhibition expressed in various types of solid tumour engages with restores 4HT activity to decrease BCL2 expression, increase programmed cell death 1 (PD-1) on immune cells and mitochondrial permeability and apoptosis (Nehra et al. is considered a crucial contributor in cancer immune 2010). Moreover, the study elucidated that caspase 8 evasion (Zhang et al. 2018) that might lead to resistance inhibitors can restore all of these effects, indicating that of treatment. NF-KB modulates caspase 8 activity to alter tamoxifen The role of NF-KB in PD-L1 expression was sensitivity. However, the precise mechanism is still demonstrated by using siRNA-mediated silencing of p65 uncleared and needs to be studied further. and p50 led to a decrease in the IFN-γ-induced expression of PD-L1 in melanoma cell lines (Gowrishankar et al. 2015). In addition, transfected melanoma cells with the NF-KB mediates anti-oestrogen resistance by mutant IκB-GFP plasmid that lack the phosphorylation blocking apoptosis and inhibiting autophagy through site, which is required for proteasomal degradation, the XBP1-NF-KB signalling cascade following with IFN-γ treatment resulted in a decrease in NF-KB is a crucial mediator for X-box binding protein 1 expression of PD-L1. This result suggest NF-KB is required (XBP1) to drive the cell-fate decisions in breast cancer cells for expression of PD-L1 (Gowrishankar et al. 2015). in response to anti-oestrogen therapy and subsequent Increasing evidence has demonstrated different development of anti-oestrogen therapeutic resistance biochemical aspects of PD-L1 regulation, including (Hu et al. 2015). XBP1 is a central effector in unfolded epigenetic modification, transcriptional regulation and

https://erc.bioscientifica.com © 2019 Society for Endocrinology https://doi.org/10.1530/ERC-19-0087 Published by Bioscientifica Ltd. Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 08:06:07PM via free access Endocrine-Related P Khongthong et al. Nuclear factor kappa B and 26:6 R376 Cancer breast cancer post-translational modification (Zhang et al. 2018). The alongside with disease progression (Mazel et al. 2015). COP9 signalosome 5 (CSN5), known as deubiquitinating Furthermore, in univariate analysis, the expression of enzymes (DUBs) has been demonstrated to stabilize PD-L1 was associated with decreased overall survival in PD-L1 at the post-transcriptional level, which results in patients with luminal B subtype (P < 0.0001) (Muenst suppression of immune surveillance (Lim et al. 2016). et al. 2014). Recently, a basket study of single agent Recently, it has been shown that upon TNF-α stimulation, pembrolizumab in patient with PD-L1-positive solid the NFκB p65 subunit binds to the CSN5 promoter to tumours, which had a cohort of patient with ER-positive/ promote its transcription, which subsequently induces its HER-2-negative breast cancer demonstrated modest but binding to PD-L1 to further deubiquitinate and stabilize durable overall response in patient with ER-positive, HER- the protein (Lim et al. 2016). 2-negative breast cancer (Rugo et al. 2018). Using the ENCODE database of transcription factors, it Collectively, PD-L1 expression is more frequently was shown that p65 directly interacts with CSN5 promoter demonstrated in breast cancer that exhibit inflamed region and promotes its transcription in response to phenotype such as TNBC as well as in patients with TNF-α treatment (Lim et al. 2016). CSN5 expression was metastatic ER-positive disease. Therefore, combination reduced in shRNA p65 knockdown cells, suggesting p65 checkpoint inhibitor with standard treatment might is required for CSN5 transcription. Increased expression be beneficial for patients with high immune response of PD-L1 was found in TNFα-treated shCTRL cell, whereas metastatic ER-positive breast cancer. the expression was abolished in TNFα-treated shCSN5 cells, indicating CSN5 as a crucial factor to stabilize PD-L1 (Lim et al. 2016). Moreover, in patient samples, it NF-KB influence the cell cycle in endocrine was shown that there was positive correlation between therapy resistance CSN5 and PD-L1 expression, with high CSN5 expression Dysregulation of the cyclin D–CDK 4/6–INK4–retino significantly associated with short overall survival Lim( blastoma (Rb) pathway regulates proliferation in breast et al. 2016). In addition, high expression of CSN5 and p65 cancer and is a key contributor to hormone therapy were associated with poor recurrence-free survival (Lim resistance (Spring et al. 2016). Activation of the cyclin et al. 2016). These findings suggest the NF-KB p65 subunit D1/CDK4/6 complex induces phosphorylation of the as a crucial transcription factor, which is required for CSN5 Rb protein, followed by destabilization of its interaction transcription, and TNFα-mediated PD-L1 stabilization, with E2F transcription factors, resulting in E2F enhanced resulting in suppressed T cell function. According to these transcription of genes that promote cell cycle progression finding, reduced PD-L1 stability, which will further allow (Lange & Yee 2011). Multiple mitogenic signalling PD-L1 poly-ubiquitination and subsequent degradation pathways have been demonstrated to influence the through the inhibition of TNF-α/p65/CSN5/PD-L1 axis increasing of the expression of cyclin D1, including PI3K/ represent a potential target to restore treatment sensitivity AKT/mTOR, MAPKs, STATs, ER and NF-KB pathways in breast cancer cells. (Lange & Yee 2011). PD-L1 expression was upregulated nearly 40% of basal subtype breast cancer, and high PD-L1 expression IKKα phosphorylates both ER and SRC3 to further was associated with poor clinical features such as high increase their activity regarding cell cycle regulation grade, high proliferation, ER-negative and HER2-enriched subtypes (Sabatier et al. 2015). Importantly, combination As previously described, oestrogen induces the formation of anti-PD-L1 and nab-paclitaxel was recently approved for of the IKKα, ER and steroid receptor co-activator (SRC) 3 patient with unresectable locally advanced or metastatic complex on the promoter region of CYCLIN D1 gene. IKKα TNBC who is tumour positive for PD-L1 (Schmid et al. is also able to phosphorylate both ER and SRC3 to further 2018). increase their transcription activity (Park et al. 2005, Although PD-L1 upregulation is not widely reported Park 2017). In addition, IKKα has been reported as a key in ER-positive breast cancer, up to 20% of primary breast kinase in response to oestrogen-induced E2F1 expression. cancer with luminal B subtype have been demonstrated IKKα siRNA treatment in MCF7 cells leads to a dramatic to overexpress PD-L1 (Muenst et al. 2014). In addition, decrease in the expression of E2F1-regulated genes that are expression of PD-L1 was found 68% in circulating breast crucial for S phase entry, including TK1, PCNA, CDC25A cancer cells from metastatic ER-positive HER2-negative and CYCLIN E (Tu et al. 2006). IKKα knocked-in cells also breast cancer, indicating increased expression of PD-L1 induce upregulation of E2F1 expression (Tu et al. 2006).

Although the mechanism of how IKKα regulates pathway is involved in endocrine resistance (Montemurro E2F1 is not clear, previous data suggests that IKKα may et al. 2013). Binding to EGF induces a kinase activity of interact with p300/CBP-associated factor (PCAF), which the homo- and heterodimers of EGFRs, leading to auto- is a potent enzyme that can acetylate E2F1 (Martinez- phosphorylation of these receptors and further activation Balbas et al. 2000). A previous study demonstrated a of downstream signalling. As a consequence of EGFR nuclear role of IKKα that may activate the expression of activation, several signalling pathways are activated, NF-KB-responsive genes upon activation of cytokines including the PI3K/Akt/mTOR pathway to control cell through phosphorylation of histone H3. In response to proliferation, and the RAS/RAF/MEK/ERK to control cell TNFα activation, IKKα forms a complex with p65 and the survival and progression. Crosstalk between growth factor co-activator of cAMP-response element-binding protein to signalling and ER has long been established. A small further acetylate-specific residues on histone H3 leading amount of ER localizes at the membrane (mbER) and to increased gene expression (Yamamoto et al. 2003). interacts with other proteins, including SRC and PI3K to Therefore, it may be suggested that IKKα associates with from the mbER complex. Upon binding to oestrogen, the PCAF to acetylate E2F1 in response to oestrogen-induced mbER complex can induce the PI3K/AKT/mTOR pathway E2F1 expression. Collectively, this evidence confirms that by enhancing the tyrosine kinase activity of EGFR and NF-KB and its kinase influence hormone resistance in activates mTOR by PI3K-mediated AKT phosphorylation. breast cancer by affecting cyclin D and E2F to promote Moreover, phosphorylated AKT can phosphorylate cell cycle progression. Aberrant IKKα/NF-KB signalling both ligand-dependent and non-ligand-dependent ER. might lead to dysregulation of CYCLIN D1 and E2F1, Collectively, crosstalk between these pathways induces resulting in the persistent activity of cell cycle machinery endocrine resistance both to tamoxifen and AI. that can promote breast cancer cell growth and resistance In contrast to ER-negative breast cancer, there is some to treatment. evidence to provide a connection between HER2 and NF-KB pathway in ER-positive MCF-7 cells. In response to ionizing radiation, overexpression of HER2 activates NF-KB and growth factor signalling in the PI3K/AKT pathway, which then promotes NF-KB response to endocrine resistance activation, resulting in upregulation of several pro-survival genes such as a mitochondrial antioxidant enzyme NF-KB and EGF pathway manganese superoxide dismutase, cyclin B1 and HER-2 Patients with ER+/HER2+ metastatic breast cancer benefit itself. Consequently, MCF-7/HER-2 cells came to be both less from endocrine therapy, compared to those with radio- and chemo-resistant (Pianetti et al. 2001). Given ER+/HER2- disease (Ballinger et al. 2018). Importantly, a the role of NF-KB as a downstream effector of HER2/PI3K/ significant amount of evidence has established the EGF AKT cascade, it might be possible that mbER indirectly

TNFα LT,CD40L Targeting upstream compounds: BCR TNFR LTβR,CD40 Ibrutinib TAK1 NIK Targeting NIK Figure 3 Targeting IKKγ/ IKKγ p NEMO:iNUBs p P Alternative approaches to target NF-KB pathway. P Targeting IKKα IKKα IKKα IKKβ IKKα or IKKβ (A)Targeting BCR-induced NF-KB signalling using p p inhibitors of Burton tyrosine kinase (BTK) such as Targeting IκBα Ub p RelB p p100 degradation IκBα Ibrutinib, results in inhibition of NF-KB p65 p50 Ub downstream signalling. (B) Targeting IKKγ or Targeting nuclear NEMO using NEMO-ubiquitin interaction localization, dimerizationand DNA inhibitors (iNUBs). (C) Targeting IκBα degradation bindingof using proteasome inhibitors such as botezomib. NF-kB (D) Targeting IKKs using selective IKKα or IKKβ inhibitors. (E) Targeting NIK using small-molecule inhibitors. (F) Targeting NF-KB activity, including p65 p50 RelB p100 inhibition of nuclear translocation, dimerization and DNA binding. A full colour version of this figure is available athttps://doi.org/10.1530/ NF-kBtargetgene ERC-19-0087.

The potential role of NF-KB as a target for endocrine Declaration of interest The authors declare that there is no conflict of interest that could be therapy resistance in breast cancer has recently been perceived as prejudicing the impartiality of this review. highlighted leading to the development of inhibitors to target the NF-KB pathway; however, this is very much still in the development stage. To date, different NF-KB Funding inhibitors have been experimentally identified and This work did not receive any specific grant from any funding agency in the provide sophisticated result (Herrington et al. 2016, Paul public, commercial or not-for-profit sector. et al. 2018). These vary from broad-spectrum inhibitor of NF-KB, including anti-inflammatory agents such as glucocorticoids, non-steroidal anti-inflammatory drugs, Acknowledgements All authors have read the journal’s policy on disclosure of potential conflicts to those that selectively inhibit each step of the pathway, of interest and have none to declare. including inhibition of NF-KB nuclear translocation, inhibitor of IκB function such as proteasome inhibitor, inhibition of IκB proteins phosphorylation (parthenolide, flavopyridol) and inhibitor of IKKs (IKKα and IKKβ). References Currently, selective inhibitors of IKKβ have been AlFakeeh A & Brezden-Masley C 2018 Overcoming endocrine resistance extensively studied and demonstrated promising in hormone receptor-positive breast cancer. Current Oncology 25 S18– outcome in pre-clinical models in cancer; however, they S27. (https://doi.org/10.3747/co.25.3752) Baldwin AS 2001 Control of oncogenesis and cancer therapy resistance still lack clinical success due to severe on-target toxicities by the transcription factor NF-kappaB. Journal of Clinical Investigation (Prescott & Cook 2018). Among other novel drugs, only 107 241–246. (https://doi.org/10.1172/JCI11991) proteasome inhibitors, bortezomib and carfizomib, have Ballinger TJ, Meier JB & Jansen VM 2018 Current landscape of targeted therapies for hormone-receptor positive, HER2 negative metastatic received recent Food and Drug Administration approval breast cancer. Frontiers in Oncology 8 308. (https://doi.org/10.3389/ for the treatment of refractory multiple myeloma fonc.2018.00308) (Herndon et al. 2013). As discussed, multiple upstream Barone I, Brusco L & Fuqua SA 2010 Estrogen receptor mutations and changes in downstream gene expression and signaling. Clinical activators and downstream effectors modulate NF-KB Cancer Research 16 2702–2708. (https://doi.org/10.1158/1078-0432. signalling; therefore, targeting alternative factors in the CCR-09-1753) pathway might be a successful strategy (Fig. 3). Baselga J, Campone M, Piccart M, Burris HA, 3rd, Rugo HS, Sahmoud T, Noguchi S, Gnant M, Pritchard KI, Lebrun F, et al. 2012 Everolimus in postmenopausal hormone-receptor-positive advanced breast cancer. New England Journal of Medicine 366 520–529. (https://doi. org/10.1056/NEJMoa1109653) Conclusion and future perspective Baum M, Buzdar A, Cuzick J, Forbes J, Houghton J, Howell A & Sahmoud T 2003 Anastrozole alone or in combination with Endocrine resistance remains an important issue in clinical tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early-stage breast cancer: results of the practice for breast cancer care. NF-KB has been confirmed ATAC (Arimidex, Tamoxifen Alone or in Combination) trial efficacy to be a crucial link between the resistance signalling and safety update analyses. Cancer 98 1802–1810. (https://doi. pathways, leading to endocrine therapy failure. However, org/10.1002/cncr.11745) Baumgarten SC & Frasor J 2012 Minireview: inflammation: an instigator given the fact that breast cancer is a heterogeneous disease of more aggressive estrogen receptor (ER) positive breast cancers. and the complexity of endocrine resistance mechanism, Molecular Endocrinology 26 360–371. (https://doi.org/10.1210/ which can influence the interaction between NF-KB and me.2011-1302) Ben-Neriah Y & Karin M 2011 Inflammation meets cancer, with ER in difference levels, more work is still needed. Future NF-kappaB as the matchmaker. Nature Immunology 12 715–723. research should integrate ‘omic’ platforms in order to (https://doi.org/10.1038/ni.2060) understanding the precise mechanism of how these Cao Y, Bonizzi G, Seagroves TN, Greten FR, Johnson R, Schmidt EV & Karin M 2001 IKKalpha provides an essential link between RANK two transcription factors interact with each other and signaling and cyclin D1 expression during mammary gland

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Received in final form 28 March 2019 Accepted 4 April 2019 Accepted Preprint published online 4 April 2019